This invention relates to the detection of biological moieties such as infectious agents, pathogens, or biomarkers. In particular, this invention relates to a nanoplasmonic platform for the detection and quantification of one or more pathogens such as HIV, HBV, or E. coli at a point-of-care (POC) using localized surface plasmon resonance (LSPR) principle.
Detection of infectious agents or pathogens (e.g., virus, bacteria, and fungi) is critical for homeland security, public and military health. In recent years, infectious diseases, especially viral outbreaks such as SARS, H1N1 and HIV, have a tremendous global healthcare impact, since such viruses could rapidly evolve, spread, and turn into pandemics such as HIV/AIDS and Spanish flu that had a devastating global impact causing totally over 80 million deaths. To identify and control forthcoming epidemics, it will be a critical to develop rapid, reliable, accurate, and sensitive diagnostic technologies that have the ability to be tailored to multiple settings.
Effective, rapid, accurate, and simple detection of complex pathogens and infectious agents still poses significant biological and engineering challenges. Among other things, biological challenges can arise due to the presence of multiple subtypes and strains of the pathogen that makes it difficult to achieve repeatable and reliable capture efficiencies from bodily samples without demanding lengthy sample preparation steps.
To provide one exemplary case of the cost of this unaddressed detection need, it is estimated that annually over 450,000 infants are infected with HIV through mother-to-child transmission (MTCT), which is the primary cause of AIDS in children. Rapid progression of AIDS in infants causes early death. Combined data from nine clinical trials in Africa showed that 35% of HIV-1 positive infants die by the age of 1 and 52% of HIV-1 infected children die by the age of 2.
Studies have shown that long-term suppression of HIV-1 replication in infants can be achieved by initiating antiretroviral therapy (ART) to reduce AIDS-related morbidity and mortality, and improve the quality of life. In order to provide early AIDS care and ART to HIV-infected infants, early diagnosis is key as ART is not provided until infection has been established.
However, simple and rapid serological assays cannot detect HIV-infected infants until 18 months after birth. The late identification of AIDS children is due to the interference of maternal HIV-1 specific antibodies, which are passively transferred through the placenta and persist in infants for approximately 18 months. Unfortunately, the viral load assays used in developed countries are expensive and require significant instrumentation. Thus, the lack of cost-effective POC viral load assays that can effectively reach patients living in rural, isolated settings prevents identifying HIV-infected infants that would greatly benefit from starting ART. Thus, the monitoring the viral load of HIV at a POC for many patients, and infants in particular, is still an unaddressed challenge. Traditional detection techniques such as culturing, enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) cannot be implemented at POC settings without equipped laboratories and extensive infrastructure.
In developed countries, HIV-1 viral load is monitored using commercial RNA assays, such as Roche COBAS®, Abbott RealTime, Siemens Versant™ and bioMerieux NucliSens®. However, implementation of these assays requires expensive equipment (e.g., thermal cyclers, $20,000), highly skilled personnel, and expensive reagents ($50-200 per test in the US), which in resource-constrained settings is unaffordable and unsustainable. Alternative viral load assays have been developed, such as the Ultrasensitive p24 assay, the ExaVir™ RT viral load assay, and real-time reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). However, these assays are still costly requiring refrigeration and skilled operators. Additionally, throughput of the ExaVir™ RT viral load assay is low, with turnaround time of two days and is limited to 180 samples per week per operator. Miniaturized conventional ELISA for detection of p24 antigen or RT-qPCR for detection of HIV-1 RNA has been developed. However, these methods require complex on-chip designs due to multi-step manipulations such as labor-intensive sample preparation (plasma separation and RNA extraction), amplification (expensive reagents) and detection. Additionally, these methods require parallel testing of external standards for a standard curve, which further increase the device complexity. Thus, current viral load assays, due to technical requirements and costs, are not available to benefit AIDS patients at the POC in resource-constrained settings.
For early identification of HIV-positive infants, isolation of HIV-1 in cell cultures was initially used, although it was time-consuming, costly and technically demanding. Most commonly, MTCT is diagnosed via PCR to amplify HIV-1 DNA integrated into the genome of white blood cells (WBCs). However, performing PCR still requires highly-trained operators, is time-consuming, and more expensive than current rapid serological assays. There have been efforts to develop miniaturized PCR chips including chips employing RT-qPCR for HIV-1 RNA detection.
Thus, there remains a generalized need for the detection and quantification of one or more infectious agents or pathogens at the POC. More specifically, there is a need for new, simple, highly sensitive, specific, accurate, reliable, rapid, and feasible viral load assays that are necessary to avoid further infectious agent and pathogen propagation and to screen for initiating early treatment at the inception of an epidemic.